Acute traumatic injury to a joint is known to increase the risk for the development of secondary osteoarthritis, but it is unclear how this process occurs. The existence of such a discrete event that can lead to an increased risk of osteoarthritis has spurred interest in developing in vitro models of traumatic joint injury. Whereas animal and human studies have focused on injuries to the intact joint, in vitro models have focused on understanding the effect of the injurious mechanical compression primarily on the articular cartilage itself. Although in vitro models cannot address all the events that occur in response to injury, they allow quantification of specific events and mechanisms involving the effects of well-defined loading regimens on cartilage. In addition to describing the effects of injurious compression on the matrix and the chondrocytes, evidence can be gathered to describe how and why the chondrocytes respond. The Specific Aims of this proposal are: (1) Utilize our newly developed in vitro injury model involving mechanically injured cartilage incubated in the presence (and absence) of cytokines (including IL-1, TNF-alpha, IL-6) to quantify the synergistic effects of injury and cytokines on chondrocyte-mediated matrix turnover; (2) Extend and utilize our newly developed in vitro injury model involving normal and mechanically injured cartilage incubated in the presence (and absence) of joint capsule tissue to quantify the synergistic effects of injury and co-culture on chondrocyte mediated catabolic and anabolic processes; (3) Quantify the synergistic effects of injury and coculture with cytokines or human joint capsule tissue on chondrocyte-mediated catabolic and anabolic processes in human knee versus ankle cartilages; (4) Quantify the molecular structure and molecular mechanical function of matrix proteoglycans and collagens synthesized by chondrocytes and lost to the medium from injured bovine and human cartilages, using molecular biophysical methods including atomic force microscopy and high resolution force spectroscopy; and (5) Determine the effects of graded levels of mechanical injury on gene expression, signaling pathways, and post-translational modifications of CS and KS-GAGs relevant to the molecular mechanics and electromechanics of aggrecan-rich ECM.